During development, embryonic muscle cells express embryo specific genes which are not expressed in adult cells. It has been postulated that embryo specific genes perform specialized functions in the morphogenesis and/or physiology of developing embryonic cells. We have characterized an embryo specific muscle gene, the gene encoding cardiac troponin T (cTNT). This gene is expressed in concert with other muscle genes during early embryonic development but transcription of this gene is repressed at day 14 of chick skeletal muscle development. Available evidence indicates that nerve probably provides the extrinsic trigger for signalling the stage specific repression of cTNT gene transcription. In order to characterize the molecular mechanisms which govern the developmentally regulated transcription of the cTNT gene we will perform transfection experiments into cultured embryonic skeletal muscle cells. Deletion and oligonucleotide directed mutagenesis will be used to modify regions affecting cTNT promoter activity, to identify the nucleotide sequence elements which act in cis to govern transcription. Regions which are essential for cTNT promoter activity will be identified, as well as regions which augment activity of the essential Promoter elements. In order to determine how nuclear proteins interact with cTNT gene regulatory sequences we will perform DNA-protein binding studies and use footprint techniques to map the nucleotide sequences involved in trans factors binding. Long term goals involve the isolation and functional testing of trans factors which regulate the cTNT gene. The mechanism of the developmentally programmed repression of the cTNT gene will be studied in a cell culture model which mimics the repression of this gene in vivo. cTNT gene constructions will be transfected into cultured skeletal muscles followed by induction of cTNT gene repression. Deletion mapping will then be used to determine the location of the cis sequences required for response to the repression signal. Once identified, these cis elements will be analyzed by site directed mutagenesis and DNA-protein binding analysis as described above. The cTNT gene affords a novel opportunity to study the Positive and negative developmental regulation of an embryo-specific muscle gene. The overall goal of these experiments is to characterize that regulation in molecular detail.